Cell-cell and cell-matrix adhesions play essential roles in endothelial barrier function. Various tetraspanins are expressed in endothelium, form tetraspanin-enriched microdomains with cell adhesion proteins such as integrins, and regulate endothelial cell adhesion and vascular permeability. Our early study revealed that tetraspanins are required for maintaining proper endothelial adhesiveness, endothelial barrier, and vascular permeability. We have demonstrated that, to sustain vascular stability, tetraspanin-enriched microdomains tune the balance of Rac1 and RhoA small GTPase activities or the balance of cortical actin meshwork and stress fibers to sustain endothelial cell-cell and cell matrix adhesions. But the in-depth mechanisms by which tetraspanins regulate endothelial barrier function still remain elusive. We will use tetraspanin CD151 as example in this study to elucidate the mechanisms by which tetraspanin-enriched microdomains regulate endothelial barrier function and vascular permeability. The overarching hypotheses of this project include that, at the cellular level, CD151 promotes endothelial barrier function by primarily increasing endothelial cell-matrix interactions, which subsequently elevates endothelial cell-cell adhesion. Meanwhile, CD151 can also directly reinforce endothelial cell-cell interaction. At the molecular level, CD151 reinforces cell adhesions by enhancing the functional accessibility and nanoscale organization of cell adhesion proteins at endothelial cell surface. Specifically, we will first unravel the mechanism by which CD151 reinforces endothelial cell- matrix adhesion. We will assess both in vitro and in vivo mechanistic roles of CD151 in integrin activation and accessibility at the basal surface of endothelial cells. Secondly, we will reveal the mechanism by which CD151 reinforces endothelial cell-cell adhesion. We will assess both in vitro and in vivo mechanistic roles of CD151 in maintaining structural and functional integrity of endothelial cell-cell contacts, which directly prevents endothelial hyper-permeability. Finally, we will delineate the signaling mechanisms by which CD151 reinforces endothelial barrier and reduces vascular hyper-permeability. Hence, the general goal of this project is to understand how CD151 sustains the endothelium barrier function by assessing both in vitro and in vivo mechanistic roles of CD151 in stabilizing the structural and functional interactions of endothelium with the underlying basement membrane and in maintaining the structural and functional integrity of endothelial cell-cell contacts and junctions. From these studies, we will understand why and how tetraspanin-enriched microdomains are important for endothelial barrier function, establish a novel paradigm between vascular permeability and membrane organization of cell adhesion molecules, and delineate the signaling axis that governs the fine balance of small GTPases in endothelium. From the in-depth mechanistic study, we will develop an integrated understanding of the unique features of tetraspanin-enriched microdomains, which will ultimately lead to the development of therapeutic mean against vascular diseases.